Figure 5.

GS inhibition by MSO impedes the differentiation of monocytes into DCs. (A-F) Human dendritic cells (DCs) were differentiated from monocytes in the presence of FCS, GM-CSF, IL-4, and with or without 2 mM glutamine (Gln) and/or 1 mM MSO for 7 days. (A) Cell count and (B) viability of differentiated DCs were determined on day 7 using the CASY Cell Counter. Symbols represent individual donors; lines link data points from the same donor. Statistical significance was calculated using one-way ANOVA and Tukey’s multiple comparison test (*p < 0.05, **p < 0.01). (C) On day 7, pictures were taken to document the morphology of DCs; one representative picture is shown for each condition. (D) The mRNA expression of GLUL by DCs was determined by qPCR and normalized to 18S mRNA. Statistical significance was calculated using Wilcoxon’s matched pair signed rank test (no significance detected). (E) Western Blot analysis of glutamine synthetase (GS) in DCs. (F) DCs were incubated with 2 mM [¹³C₅]glutamate in the presence or absence of 1 mM MSO for 24 h. After 24 h, cells were lysed and mean enrichment of ¹³C glutamate and glutamine was determined in cell pellets. Shown are median values and single data points. Statistical significance was calculated using Wilcoxon’s matched pair signed rank test (no significance detected). (G-J) THP-1 cells were cultured in the presence or absence of 2 mM glutamine (Gln) and/or 1 mM MSO. Cell yield (G) and cell viability (H) of human THP-1 cells were analyzed after 48 h. Shown are median values and single data points. Statistical significance was calculated using one-way ANOVA and Tukey’s multiple comparison test (*p < 0.05, **p < 0.01). (I) After 48 h, pictures were taken to document the morphology of THP-1 cells; one representative picture is shown for each condition. (J) Oxygen consumption was monitored in the absence or presence of 2 mM Gln or 2 mM Glu and/or 1 mM MSO using the PreSens technology (mean values, n = 3).